Favipiravir, an antiviral drug, in combination with tamoxifen exerts synergistic effect in tamoxifen-resistant breast cancer cells via hTERT inhibition

Tamoxifen (TAM) is one of the most successful treatments for breast cancer; however, TAM resistance continues to be a significant barrier. TAM resistance has been reported to be associated with increased expression of human telomerase reverse transcriptase (hTERT). This enzyme shares structural similarity with RNA-dependent RNA polymerase (RdRp) enzyme of RNA viruses, suggesting that RdRp inhibitors may also inhibit hTERT. Favipiravir (FAV) is an antiviral drug that inhibits RdRp of RNA viruses. Thus, we propose that FAV may also elicit an antitumor effect by suppressing hTERT. This study aimed to investigate the effect of FAV and TAM on TAM-resistant breast cancer (TAMR-1). The cell viabilities were determined. The levels of CDK1/ hTERT, in addition to regulators of hTERT-targeted signaling pathways were measured. Apoptosis, migration, and cell cycle distribution were also determined. Our data revealed that the combination of TAM and FAV suppressed cell proliferation synergistically (CI < 1) and resulted in a significant change in cell migration and apoptosis. Indeed, this was associated with reduced levels of hTERT and CDK1 and shift in the cell cycle distribution. Our findings suggest that the TAM/FAV combination exhibits synergistic effects against TAMR-1 human breast cancer cells by targeting hTERT.


Tamoxifen and favipiravir monotherapy decreased the viability of MCF-7 and TAMR-1 cancer cells while cotreatment potentiated the effects
As a first step, the viability of the carcinoma cell lines in the presence of TAM or FAV or their combination was determined using sulforhodamine B (SRB) assay.It was observed that FAV and TAM had an inhibitory effect on MCF-7 and TAMR-1 breast cancer cells in a dose-dependent manner following 48 h of drug incubation.The average IC50 values of TAM and FAV in MCF-7 cells were 4.5 μM and 40 μM, respectively (Fig. 1a,b), whereas the average IC50 values of TAM and FAV in TAMR-1 cells were 51 μM and 40 μM, respectively (Fig. 1c,d).Although MCF-7 cells are more sensitive to TAM monotherapy than TAMR-1 cells, both cell lines showed the same sensitivity toward FAV with equal IC50 values of 40 μM.
In both cell lines, there was a significant decrease in viability in all combination-treated samples as compared to monotherapy (Fig. 1e,f).When TAM (50 μM) and FAV (50 μM) were combined to treat TAMR-1 cells, the inhibitory rate reached 78%, whereas treatment with TAM alone achieved an inhibitory rate of 48%.In MCF-7 cells, treatment with TAM (2.25 μM) in combination with FAV (100 μM) inhibited cell growth by approximately 16% when compared to TAM alone.
CompuSyn software was used to analyze the effect of the combination's interaction to see whether combinations would have a synergistic effect and result in a greater reduction in cell growth than the single drugs.For both drugs, synergism equivalent to a combination index (CI) of < 1 invariably resulted in a favorable dose-reduction index of > 1 (see Supplementary Fig.*** S1 online).As a result, for the subsequent studies using TAMR-1 cells, a 50 μM FAV and 50 μM TAM combination regimen was chosen since it had the lowest CI (0.75) when compared to other combinations.

The effect of TAM (50 μM), FAV (50 μM), or their combination on the gene expression of hTERT and CDK1 in TAMR-1 cells
To investigate the putative suppressive role of FAV on hTERT expression and consequent synergistic effect in TAMR-1 cells, the gene expression levels of hTERT and CDK1 were assessed after treating cells with TAM or FAV or their combination.The gene expression levels of hTERT and CDK1 were found to be significantly higher in TAMR-1 cells than in MCF-7 cells (P < 0.0001) (Fig. 2), indicating that the upregulation of these proteins correlates with TAM resistance.Each drug and their combination significantly decreased hTERT and CDK1 expression when compared to the TAMR-1 control (P < 0.0001).Moreover, the combination significantly decreased the hTERT and CDK1 gene expression levels when compared to TAM monotherapy.

The effect of TAM (50 μM), FAV (50 μM), or their combination on wound healing in TAMR-1 cells
The ability of TAMR-1 cells to migrate was examined using a wound healing test to see if FAV may improve TAM treatment and prevent TAMR-1 cell migration.Figure 4a illustrates TAMR-1 control cells at 0 h.When compared to the control at 48 h (Fig. 4b), TAM monotherapy significantly reduced migratory capability (Fig. 4c), while FAV monotherapy (Fig. 4d) had no significant effect on the wound healing ability of TAMR-1 cells (P > 0.05).Interestingly, the combination of FAV (50 μM) and TAM (50 μM) resulted in a substantial delay in wound healing when compared to FAV and TAM monotherapies (P < 0.0001) (Fig. 4e).To investigate whether TAMR-1 cells undergo apoptosis upon treatment with tamoxifen, favipiravir, or their combination, cell staining with annexin V and propidium iodide (PI) was performed and cells were analyzed using flow cytometry.Induction of cell apoptosis of TAMR-1 cells treated with TAM (50 μM) or FAV (50 μM) monotherapy or their combination was observed at 48 h (Fig. 5).In comparison to the single-drug treatment groups, the number of late apoptotic and necrotic cells in the combination-treatment group was increased.The percentage of TAMR-1 cells that were found to be necrotic or apoptotic is shown in Fig. 5b.To confirm that combination therapy induces apoptosis in TAMR-1 cells, the levels of the proapoptotic protein, BAX, and the antiapoptotic protein, BCL-2, were measured (Fig. 5c).The Western blot results revealed that the expression level of BAX was significantly increased, while the expression of BCl-2 was decreased after treatment with the drug combination (P < 0.05).These outcomes matched the outcomes of the SRB test, suggesting that the addition of FAV to TAM has a synergistic effect on TAMR-1 cells.

The effect of TAM (50 μM), FAV (50 μM), or their combination on cell cycle distribution of TAMR-1 cells
The cell cycle distribution was assessed by flow cytometry after 48 h to further determine if the antiproliferative impact caused by TAM, FAV, or their combination is linked to cell cycle arrest.Figure 6 shows that there was a clear alteration in the distribution of different phases when cells were treated with TAM, FAV, or their combination.Remarkably, TAM arrested the cell cycle at the S (58.29%) and G 2 /M (4.40%) phases, while FAV arrested the cell cycle at the G 0 /G 1 phase (97.06%).The combination of TAM and FAV significantly increased the percentage of cells accumulated at the G 2 /M phase (9.36%) compared to the control (0.28%) and each drug alone (TAM, 4.4%; FAV, 0.07%).When compared to the control group, TAM-treated cells showed a significant increase in cyclin D1 (CCND1) levels, while FAV treatment significantly decreased the CCND1 level.Interestingly, combining the drugs significantly increased the CCND1 level compared to the control but CCND1 showed a significant decrease compared to its corresponding level in TAM-treated cells.Moreover, the combination significantly decreased cyclin B1 (CCNB1) levels compared to the control and to each drug alone.

Discussion
Breast cancer is the most frequent cancer in females and the main cause of death from cancer 2 .TAM is one of the most chemotherapeutic medications used to treat breast cancer, however with time, cancer cells become resistant to TAM [8][9][10][11] .Some genes have been found to be associated with mechanisms of tamoxifen resistance 39 .Thus, exploration of more effective strategies is demanded to overcome resistance in breast cancer cells, improve TAM efficacy, and decrease undesirable side effects 9,[38][39][40] .Diverse attempts are being investigated to prevail this impediment including novel therapeutic agents, immunotherapy, gene therapy, and combination therapy 10,40,41,45 .Nonetheless, clinical outcomes are still mediocre urging a thorough understanding of the underlying chemoresistance mechanisms, and improvement of treatment approaches.
A known resistance mechanism focuses on hTERT overexpression 16,17 .hTERT promotes cancer cell proliferation, angiogenesis, migration, and metastasis 39,40 .Elevated hTERT levels have been related to poor prognoses in solid tumors, including gastric, cervical, lung and breast cancer 48 .hTERT has previously been shown to have RdRp activity, which is required for tumor development 42,43 .
FAV, an RdRp inhibitor, is used against various RNA virus infections.In this study, FAV was combined with TAM due to its RdRp inhibitory effects and the structural similarity between hTERT and RdRp 44,45 .In addition, infection is more common in patients with cancer than in the normal population 46,47 .It has been reported that knockdown of hTERT inhibits cancer proliferation and attenuates resistance to radio-and chemotherapy 16,48 .Moreover, the phosphorylation of hTERT by CDK1 participates in cancer development 31 .CDK1 is crucial in cell cycle control and is considered as a key element of mitosis 56 .CDK1 inhibitors is an important target for cancer treatment and are already involved in some clinical trials 51,52 .Also inhibiting CDK1 has been reported to induce apoptosis selectively in MYC-dependent breast cancer cells 59 .According to our gene expression analysis, TAMR-1 cells had higher levels of hTERT and CDK1 than MCF-7 cells.Meanwhile, treatment with TAM, FAV, and their  www.nature.com/scientificreports/reduced the expression of CDK1 and hTERT.Interestingly, the combination restored hTERT and CDK-1 levels in TAMR-1 cells to those in MCF-7 cells.These findings are in agreement with a prior report stating that the downregulation of hTERT in MCF-7 cells rendered cells more sensitive to chemotherapeutic drugs 17 .Combining metformin and silibinin inhibited T47D breast cancer cells' proliferation synergistically via inhibition of hTERT 60 .Moreover, a combination therapy of roscovitine, a pan-CDK-1 inhibitor, followed by doxorubicin enhanced doxorubicin efficiency and reduced toxicity in triple-negative breast cancer 61 .
The possible FAV-induced mechanisms underlying the downregulation of hTERT expression and synergistic potentiation of breast cancer cell death was also investigated in this study.hTERT was found to be regulated by wingless (Wnt), mammalian target of the rapamycin (mTOR), and mitogen-activated protein kinase (MAPK) signalling pathways 54,55 .TNF-α controls protein synthesis by activating MAPK and regulates telomerase activity by translocating hTERT protein coupled to NF-B p65 from the cytoplasm to the nucleus, a substantial step for inducing telomere elongation 33 .MAPK then causes the eukaryotic initiation factor, eIF4E, to be phosphorylated.Moreover, TNF-α regulates the mTOR pathway as assessed by the phosphorylation status of the mTOR substrate, 4E-BP1 33,34 .mTOR mediates the phosphorylation of 4E-BP1, leading to the dissociation and release of eIF4E from 4E-BP1, enabling eIF4E to promote cap-dependent translation, resulting in increased protein synthesis of oncogenic mRNA 36 .Dependent on the tumor microenvironment, 4E-BP1 plays a regulatory role by selectively modulating the translation of particular transcripts that function as drivers of cancer cell proliferation and progression by adapting the tumor to metabolic and genotoxic stress 64 .
Moreover, Zhang et al. 65 reported that high levels of TNF-α promote chemoresistance in breast cancer cells.4E-BP1 has also been linked with poor prognoses and drug resistance in patients with cancer.Knockdown of 4E-BP1 in breast cancer cells has been found to result in substantial decreases in cell proliferation 66 .eIF4E is a known protumorigenic factor, with elevated expression or activity in a variety of malignancies.High expression of eIF4E indicates poorer recurrence-free survival levels 67 .Moreover, CDK1 was recently reported to phosphorylate 4E-BPI in the absence (or low levels) of mTOR kinase as an alternative method to maintain phosphorylation during mitosis 68 .Our data revealed that TAMR-1 cells showed significantly higher levels of TNF-α, eIF4E, and 4E-BP1 in comparison to MCF-7 cells.Moreover, the combination of TAM and FAV significantly lowered the levels of TNF-α, eIF4E, and 4E-BP1 when compared to each drug alone.
Our study demonstrates that a combination of TAM and FAV can synergistically inhibit cell proliferation and increase apoptosis in MCF-7 and TAMR-1 cells.TAM-treated TAMR-1 cells showed a higher IC50 than TAM-treated MCF-7 cells, whereas FAV showed a consistent IC50 across both cell lines.These results are in agreement with prior reports that demonstrated that TAM treatment inhibited cell growth and apoptosis in MCF-7 cells, and that breast tumors developed resistance to TAM 8,60 .Furthermore, it has been reported that FAV has an inhibitory effect on A549 lung cancer cells 72 .Our results showed that the percentage of apoptotic and necrotic cells in the combination-treated group were increased compared with the single-drug treatment group.TAM has been reported to induce apoptosis in MCF-7 cells 3 .Moreover, downregulating hTERT results in the induction of apoptosis and the suppression of cell viability in MCF-7 cells 73 .Two apoptotic pathways exist: the extrinsic and intrinsic pathways.The intrinsic pathway, also known as the mitochondrial pathway, is controlled by BCL-2 family proteins, including the proapoptotic protein, BAX, and the antiapoptotic protein, BCL-2.In cancer cells, BCL-2 is usually upregulated, inhibiting the proapoptotic BAX; therefore, inhibiting apoptosis 63,64 .Our data demonstrate that using a combined therapy of TAM and FAV promotes downregulation of BCL-2 antiapoptotic proteins and the upregulation of BAX proapoptotic proteins in TAMR-1 cells.The increased expression of BAX protein, associated with BCL-2 decrease, in cells incubated with combined doses of FAV and TAM illustrates that combination therapy ameliorates the BAX effect, leading to the induction of the intrinsic apoptosis pathway.Similarly, a previous study showed that combining TAM with histone deacetylase inhibitors in TAMR-1 cells induced apoptosis by downregulating BCL-2, suggesting that a high level of Bcl-2 is a key driver of TAM resistance 62 .
The distribution of the TAMR-1 cells in the cell cycle was determined for a better understanding of the mechanisms of TAM and FAV underlying cell growth inhibition.The analysis of flow cytometry data revealed that TAM and its combination with FAV caused cells to assemble at the S and G 2 /M phases, while simultaneously depleting G 0 -G 1 -phase cells.Conversely, FAV triggered cells to aggregate at the G 0 -G 1 phase.The drugs inhibited the growth of MCF-7 breast cancer cells by delaying the cell cycle transition.However, different studies have found that TAM causes MCF-7 cells' cell cycle to stop at the G0-G1 stage 65,66 .Western blot analysis of CCND1 and CCNB1 showed that the drug combination led to a significant increase in CCND1 when compared to the control group, while its level was lower than in TAM-treated cells.It was previously reported that CCND1 overexpression is associated with better outcomes for patients with breast cancer but its overexpression is linked to TAM resistance 78,79 .CCND1 phosphorylates and inactivates retinoblastoma protein, allowing cells to progress from G1 phase to S phase 79 .High CCNB1 cyclin B1 expression is associated with poor survival in breast cancer 80 .This agrees with our results, which showed that the combination significantly decreased its levels.
Our data demonstrated a significant reduction in cell migration of TAMR-1 cells when combination treatment of TAM and FAV was implemented compared to each drug alone.This result was in accordance with a previous study that showed a slowdown in wound healing by TAM in MCF-7 cells in a dose-dependent manner 81 .Moreover, it was previously reported that hTERT increases cell migration and its regulation with miR-138-5p suppresses cell growth and migration [82][83][84] .
In conclusion, hTERT overexpression is a possible mechanism contributing to TAM therapy resistance.Our findings confirmed that FAV synergistically enhances the anticancer effect of TAM in TAMR-1 cells by downregulating hTERT, TNF-α, eIF4E, and 4E-BP1 expression.The combination of TAM and FAV elicits inhibition of proliferation and invasiveness as well as inducing apoptosis and a shift in the cell cycle distribution (Fig. 7).Thus, our results identified a new mechanism of action for FAV in TAM-resistant breast cancer cells, suggesting that combining FAV with TAM might be an effective therapeutic option to overcome endocrine resistance in

Sulforhodamine-B (SRB) (cytotoxicity) assay
MCF-7 and TAMR-1 cells were seeded at a concentration of 2 × 10 3 cells/well in 96-well plates with RPMI-1640 media, 10% FBS, and antibiotics.Plates were maintained at 37 °C in a 5% CO 2 atmosphere overnight.Cells were then treated with different concentrations of TAM or FAV (0-200 μM) and incubated for 48 h.IC50 values were generated using GraphPad Prism 8.4.2.The combination regimen was designed using the IC50 and half IC50 of TAM and increasing doses of FAV (0-200 μM).The SRB assay was used to perform the cytotoxicity test.The optical density for each well was determined using ELISA microplate reader (Sunrise, Tecan, Germany).Control cells were incubated without the drug.Using CompuSyn software, we employed the CI approach 85 to examine if the cytotoxic interactions of TAM and FAV were synergistic, additive, or antagonistic.

hTERT and CDK-1 gene expression
Total RNA was extracted from treatment and control samples using a total RNA purification kit (Jena Bioscience, Munich, Germany).A cDNA archive kit (Applied Biosystems, Foster City, California, USA) was used to transform RNA to complementary DNA.GoTaq PCR master mix (Promega Co., Madison, USA) was used for qPCR, which included 25 µL of master mix, 1 µL of forward and reverse primers, 1 µL of cDNA, and 0.25 µL of CXR reference dye then completed to a final volume of 50 µL.All analyses were conducted in triplicates on a 7500 qPCR system (Applied Biosystems, Foster City, CA, USA).The 2 −ΔΔCt method was employed for data www.nature.com/scientificreports/analysis.Outcome results were recorded as relative expression levels after being adjusted to GAPDH 86 .The primer sequences used were recorded in Table 1.

TNF-α, elF4E, and 4E-BP1 levels
The TNF-α level was measured in cell culture supernatants, while the 4E-BP1 and eIF4E protein levels were measured in the cell lysate.The TNF-α, 4E-BP1, and eIF4E levels in MCF-7 and TAMR-1 cells were estimated using human TNF-α ELISA kits (ab285312, Abcam, UK), human eIF4eBP1 ELISA kits (ab289651, Abcam), and human eIF4E ELISA kits (ab214564, Abcam), respectively.An ELISA reader (TP-Thermoplate Reader) was used to measure each parameter three times, following the manufacturer's directions, at 450 nm.Total protein was measured, and the results were expressed as mean concentration per 1 mg of total protein ± standard deviation.

Cell migration
TAMR-1 cells were seeded onto a 6-well plate.Using a sterile pipette, a single scratch was made creating a cellfree area, then the plate was rinsed to remove excess cells.Afterwards, the medium in the wells was aspirated and replaced with medium containing TAM or FAV or their combination.Controls comprised untreated wells at 0 and 48 h.An inverted microscope (DFC290, Leica, Wetzlar, Germany) was used to capture the images to analyze the size of the formed gap for each well.

Annexin V-FITC/PI for detection of apoptosis by flow cytometry
Flow cytometry was performed to examine apoptosis and necrosis of human cancer cells.A FITC Annexin V kit was used to further investigate apoptosis.Before being washed with cold PBS, all aspirates were centrifuged for 5 min at 13,000 rpm at 4 °C.The cells were extracted and centrifuged for 5 min at 2500 rpm at 10 °C.The supernatant was discarded, and the pellet was rinsed with 1 mL PBS before centrifugation at 2500 rpm for 5 min at 10 °C.Pellets were incubated for 30 min in PBS (50 μL) with Annexin V-FITC and PI.After incubation, flow cytometry analysis was carried out in 300 μL of PBS containing incubated cells in a flow cytometer (BD Biosciences LSR II FACS, New Jersey, USA).Apoptotic cells release phosphatidylserine extracellularly, which is detectable by using annexin V-labeled fluorescence.TAM or FAV were added to TAMR-1 cells alone and in combination, with PI antibodies and annexin V. Early apoptotic cells express annexin + PI−, late apoptotic cells expressed annexin + PI+ and necrotic cells expressed annexin-PI+.

Cell cycle analysis
Flow cytometry was utilized to assess the cell cycle in TAMR-1 cells, which were treated with FAV or TAM or their combination.After treatment, cells were trypsinized and fixed using 70% (v/v) ethanol and left overnight at 4 °C.After being rinsed twice with PBS, fixed cells were resuspended in PBS (Beyotime, Jiangsu, China) containing 50 g/mL PI and 0.1 mg/mL RNaseA.Following 30 min of incubation at 37 °C in the dark, the cells were analyzed using a flow cytometer (BD Biosciences LSR II FACS, New Jersey, USA).The cell count was estimated in each stage of the cell cycle (G0/G1, S, and G2/M).

Western blot assay
Ice-cold lysis buffer (1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl; pH 7.4) was added to the cells to obtain the cell lysate required.To separate the protein from the cell lysate, the proteins were subjected to electrophoresis on 12% SDS-polyacrylamide gels and electrophoretically blotted to Amersham Hybond P Western blotting membranes (GE10600021 Sigma, Sigma-Aldrich, MO, USA).After blocking with 5% skim milk, the membranes were probed with primary antibodies mouse anti-β-actin and CCND1 or CCNB1 or BAX or BCL-2 monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, California, USA) for 1 h followed by HRP-conjugated rabbit antimouse IgG or HRP-conjugated goat antirabbit IgG.Quantification of the protein-bound bands was performed by image analysis using the ChemiDoc MP imaging system (v3, Hercules, California, USA).

Figure 1 .
Figure 1.The surviving fractions of MCF-7 and TAMR-1 cells with different concentrations of TAM (a,b) or FAV (c,d), or their combination (e,f), respectively, for 48 h.The values shown are the mean ± SD of two independent experiments performed in triplicate.The surviving fraction is the percentage of viable cells in comparison to the control.Surviving fraction = optical density (treated cells)/optical density (untreated cells).

Figure 2 .
Figure 2. qPCR analysis for the expression levels of CDK1 (a) and hTERT (b) in MCF-7 and TAMR-1 cells.Data represented in the bar graphs are from three independent experiments.The statistical differences were investigated using a one-way ANOVA test followed by a Games Howell posthoc test.***Significant at P < 0.0001.****Significant at P < 0.00001.

Figure 3 .
Figure 3. TNF-α (a), eIF4E (b), and 4E-BP1 (c) levels in MCF-7 and TAMR-1 cells.Data represented in the bar graphs are from three independent experiments.One-way ANOVA test followed by Games Howell post hoc test was used to examine statistical differences.**Significant at P < 0.001.***Significant at P < 0.0001.

Figure 4 .Figure 5 .Figure 6 .
Figure 4. Wound healing assay of the treated TAMR-1 cells.Panel (a) represents the control at 0 h, (b) represents the control at 48 h.(c-e) represent TAM, FAV, and their combination at 48 h, respectively, at concentrations of 50 μM each.Panel (f) shows the bar graph quantifying the migration of the cells for each drug.Plotted points are the mean representation of the data ± SD.The statistical differences were investigated using a one-way ANOVA test followed by a Games Howell post hoc test.***Significant at P < 0.0001.

Figure 7 .
Figure 7. Graphical abstract displaying the role of tamoxifen and Favipiravir combination on TAMR-1 breast cancer cells' proliferation and angiogenesis.